Early prenatal diagnosis to detect fetal genetic disorders is desirable for both expectant mothers and physicians to make informed decisions. Until recently, non-invasive screening to identify an individual's risk for fetal aneuploidy (predominantly trisomy 21) relied on either maternal serum analytes and/or ultrasonography at gestational weeks 10 to 13 or 16 to 18 weeks (or at both points) with detection rates of 70-96% and a false positive rate of ˜5%, depending on the screening method employed. A positive screening result requires a subsequent invasive procedure (amniocentesis or chorionic villus sampling (CVS)) for confirmatory definitive diagnosis, which carries a small, but significant risk of miscarriage and the results are rarely available before 13 weeks of pregnancy because of the time required for cell culture and analysis.
During the past two decades, many laboratories around the world have attempted to develop a non-invasive prenatal test for the diagnosis of chromosomal abnormalities using rare fetal cells present in the maternal circulation. Reliably isolating sufficient fetal cells from maternal blood for genetic testing has proven difficult due to their rarity (one fetal cell per 105-108 nucleated maternal cells or 1-10 fetal cells per ml of maternal blood), the inefficiency of cell sorting techniques using fetal-cell specific markers, variable levels of maternal contamination, the tendency for fetal deoxyribonucleic acid (DNA) to disintegrate during chromosome extraction and their persistence in the maternal circulation from previous pregnancies. This approach has been abandoned by most groups for the above mentioned reasons.
It was not until 1997 that researchers discovered the existence of cell free fetal DNA (cffDNA) in the maternal circulation which presented a new possible target for non-invasive prenatal testing (NIPT). CffDNA in the maternal circulation is theorized to be derived from placental cells undergoing apoptosis (fetal DNA is cleaved into small 150-200 base pair fragments) and can be detected reliably after the seventh week of gestation. Current prenatal screening programs have now incorporated cffDNA analysis as part of prenatal management for sex determination of sex linked disorders and Rhesus D incompatibility. More recently, cffDNA has been used for aneuploidy testing (Trisomy 21, Trisomy 18, Trisomy 13, and sex chromosome aneuploidies), paternity testing and for the prediction of many pregnancy complications, including preterm birth, pre-eclampsia and fetal growth restriction. However, further research in this area has been hindered by the low concentration of circulatory fetal DNA in the maternal circulation (accounting for 10% of all cell free DNA at 11-13 weeks gestation (interquartile range 7.8-13%), the interference of excessive amounts of maternal DNA in the plasma sample, and the influence of maternal weight on fetal fraction (decreases from 11.7% at 60 kg to 3.9% at 160 kg). If the fetal fraction is below 4%, non-invasive prenatal testing is currently unable to provide a result for aneuploidy testing.
In light of this, and given that cffDNA in the maternal circulation are believed to be derived from apoptotic trophoblast cells it is hypothesized that transcervical samples may provide an alternative and perhaps superior source of both cffDNA and/or fetal cells to samples derived from maternal circulation. Unlike maternal blood in which multiple circulating fetal cell types exist, fetal cells in the transcervical samples are all of placental origin and are overwhelmingly trophoblasts.
It was long assumed that the cervical canal contained trophoblasts of fetal origin. The early embryo is covered with chorion levae, but later in the gestation the chorionic surface is smooth. However, it was not until 1971 that the presence of fetal cells in the endocervix was confirmed by identification of Y-chromosome bearing cells in midcervical mucus samples collected with a cotton swab. Subsequent reports assumed that these fetal cells were shed from the regressing chorionic villous into the lower uterine pole. In this scenario, it is most likely to occur between 7 and 13 weeks gestation, before fusion of the deciduas basalis and parietalis. Desquamated trophoblasts are believed first to accumulate behind the cervical mucus at the level of the internal opening section and then become ensconced in the cervical mucus.
These biologic events thus define the window of opportunity for endocervical sampling to be of use for prenatal diagnoses, although several studies have demonstrated trophoblast recovery as early as 5 weeks gestation.
Efforts to extract trophoblasts were first made in the 1970's. Rhine et al. (1977) described “antenatal cell extractors” that flush the endocervical canal with sterile saline to recover fetal cells. After culture, fetal metaphases from recovered cells were detected in approximately 50% of cases. However, other investigators reported negative results, leading to overall skepticism concerning clinical application. In hindsight, inability to detect fetal cells probably also reflected deficiencies in the clinicians' techniques in obtaining the endocervical specimen, as well as poor sensitivity of methods used to confirm the presence of fetal cells.
Interest was rekindled in the 1990's, several groups since have attempted to isolate fetal cells/DNA from transcervical samples using various sampling techniques including cotton swabs, cytology brushes, aspiration of mucus, and lavage of the endocervical canal or intrauterine cavity with reported detection rates of 24%-96%. Again, however, interest waned in most centres because analysis was difficult. The presumptive fetal cells embedded in mucus were not readily amenable to fluorescent in situ hybridization (FISH). More recently, molecular polymerase chain reaction (PCR) techniques for micromanipulated cell clumps of trophoblastic origin were demonstrated to have utility for transcervical samples.
Most transcervical specimens contain a variety of maternally derived cells (leukocytes, macrophages, squamous epithelia, columnar epithelia, and endocervical cells) as well as different fetal-derived cells (extravillous cytotrophoblasts, intravillous cytotrophoblasts and syncytiotrophoblasts) and free fetal/maternal nuclei. The frequency of each fetal cell type is variable and seemingly dependent on the collection method and gestational age.
It is desired to address or ameliorate one or more shortcomings or disadvantages associated with prior systems for obtaining biological samples, or to at least provide a useful alternative thereto.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or a group of elements, integers or steps, but not the exclusion of any other elements, integer or step, or group of elements, integers or steps.